Nucleic acid molecules encoding red and green emitting luciferases

ABSTRACT

Isolated nucleic acid molecules which code for luciferases able to produce the green bioluminescence of  Phrixotrhix vivianii  and red bioluminescence of  Phrixothrix hirtus  are described. The nucleic acid molecules and the luciferases encoded thereby can be used in applications such as diagnostic methods and molecular biology tools.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/993,874 nowU.S. Pat. No. 6,962,986, filed Nov. 14, 2001, which is a continuation ofapplication Ser. No. 09/516, 958, filed on Mar. 1, 2000, now abandoned,which is a continuation-in-part of application Ser. No. 09/388,290,filed on Sep. 1, 1999, now abandoned, which claims priority to Italianapplication FI98A000195, filed Sep. 2, 1998. The foregoing applicationsare incorporated by reference herein.

BACKGROUND OF THE INVENTION

Bioluminescence in beetles is characterized by a widerange of colors.Fireflies (Lampyridae) emit in the green-yellow region of the spectrum(1, 2), click-beetles (Elateridae) emit in the green-orange (2, 3), butrailroad-worms (Phengodidae) span the widest range of the spectrum, thatis, from the green to the red region (4, 5). The emission of green-redlight was suggested to be an adaptation to optimize the detection ofbioluminescence in distinct photic environments and for differentbiological functions (6). In all cases, such distinct colors arise fromstructurally homologous luciferases, which catalyze the sameATP-dependent oxidation of D-luciferin (7). Most studies about thestructure and function of beetle luciferases have focused on a set ofluciferases arising mainly from fireflies (8-14), two click-beetlespecies (15, 16), and recently a North American species of Phengodes(17). Three main factors at the level of the luciferase active site arebelieved to govern bioluminescence colors (7): (I) the presence of basicresidues catalyzing tautomerization between a ketonic (red lightemitter) and enolic (yellow-green light emitter) species of excitedoxy-luciferin (18-20); (II) the hydrophobicity of the active site (21,22); and (III) the active site conformation which affects rotation ofexcited oxyluciferin along the C₂-C₂′ bond (23). These factors may acttogether or independently to determine distinct bioluminescence colorsin luciferases of different species. The construction of chimericproteins using click-beetle luciferases (24) and firefly luciferases(25), along with mutagenesis studies (26-28), have revealed importantregions and key residues for the bioluminescence color determination.The crystallographic structure of firefly luciferase has been recentlyresolved in the absence of the substrates (29), which shows a mainN-terminal domain and a smaller C-terminal cleft which supposedly comecloser to sandwich the substrates during catalysis. Despite all thesestudies, no structural investigations had been conducted on naturallyoccurring red light-emitting luciferases.

The beautiful and rare Phrixothrix railroad-worms are probably the mostspectacular luminescent beetles, because in addition to theiryellow-green bioluminescence (λ_(max)=542-574 nm), displayed by two setsof 11 dorsal-lateral lanterns along the body, they emit redbioluminescence (λ_(max)=609-638 nm) through cephalic and postcephalicorgans (4, 5), a unique property among terrestrial creatures. Thefunction of the lateral lantern bioluminescence is probably associatedwith defensive and sexual attraction purposes, whereas in the case ofthe red lantern bioluminescence was associated with self-illumination(5); however experimental evidence is still lacking. Due to theirscarcity, only preliminary biochemical studies have been conducted aboutthese creature luciferases (4, 20). Railroad-worm and click-beetleluciferases share a common feature: they do not suffer batchromic shiftupon decreasing pH as lampyrid luciferases do (20). Due to theirpeculiar spectral properties, Phrixothrix luciferases constitute veryimportant models for understanding the mechanism of color modulation inbeetle bioluminescence.

SUMMARY OF THE INVENTION

The invention describes the cloning of the cDNAs arising from thebeetles Phrixothrix vivianii and Phrixothrix hirtus, which code theluciferases that catalyze the production of green and red light,respectively. These cDNAs were characterized by their respectivesequences of SEQ ID NOs: 1 and 3, along with their deduced amino acidsequences of SEQ ID NOs: 2 and 4, respectively.

Thus, the present invention relates to an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:1 and SEQ ID NO:3 and the complement of SEQ ID NO:1 and SEQ IDNO:3. The invention further relates to a nucleic acid molecule whichhybridizes under high stringency conditions to a nucleotide sequenceselected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3 andthe complement of SEQ ID NO:1 and SEQ ID NO:3.

The invention also relates to a vector comprising an isolated nucleicacid molecule of the invention operatively linked to a regulatorysequence, as well as to a recombinant host cell comprising the vector.The invention also provides a method for preparing a polypeptide encodedby an isolated nucleic acid molecule, comprising culturing a recombinanthost cell (e.g., bacterial, fungal, plant, insect and mammalian cells)of the invention under conditions suitable for expression of saidnucleic acid molecule.

The invention further provides an isolated polypeptide encoded byisolated nucleic acid molecules of the invention. In a particularembodiment, the polypeptide comprises the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4. The invention also relates to an isolatedpolypeptide comprising an amino acid sequence which is greater thanabout 80 percent identical and more specifically 90 percent identical tothe amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

The invention also relates to an antibody, or an antigen-bindingfragment thereof, which selectively binds to the polypeptides of theinvention, as well as to a method for assaying the presence of apolypeptide encoded by an isolated nucleic acid molecule of theinvention in a sample, comprising contacting said sample with anantibody which specifically binds to the encoded polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an in vitro bioluminescence spectra elicited by Phriothrixrailroad-worms recombinant luciferases: (A) Pv_(GR) and (B) Ph_(RE). TheBL reactions were conducted on 0.1 M Tris-HCl buffer, pH 8.0. Thesespectra were corrected for the spectral photosensitivity of theequipment and normalized.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Phrixothrix railroad-worms emit yellow-green light through 11 pairs oflateral lanterns along the body and red light through two cephaliclanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrixvivianii, which emit green light (λ_(max)=542 nm), and for the headlanterns of P. hirtus, which emit the most red-shifted bioluminescence(λ_(max)=628 nm) among luminescent beetles, were cloned. Positive cloneswhich emitted green (Pv_(GR): λ_(max)=549 nm) and red (Ph_(RE):λ_(max)=622 nm) bioluminescence were isolated. The lucifereases coded byPv_(GR) (545 amino acid residues) and Ph_(RE) (546 amino acid residues)cDNAs share 71% identity. Pv_(GR) and Ph_(RE) luciferases showed 50-55%and 46-49% identity with firefly luciferases, respectively, and 47-49%with click-beetle luciferases. Ph_(RE) luciferase has some uniqueresidues which replace invariant residues in other beetle luciferases.The additional residue Arg 352 in Ph_(RE), which is deleted in Pv_(GR)polypeptide, seems to be another important structural feature associatedwith red light production. As in the case of other railroad-worms andclick-beetle luciferases studied, Phrixothrix luciferases do not undergothe typical red shift suffered by firefly luciferases upon decreasingpH, a property which might be related to the many amino acid residuesshared in common between railroad-worm and click-beetle luciferase.

The complete cDNA nucleotidic sequences and the respective amino-acidsequences are shown in the universal genetic code. The region of thecDNA sequences which code for the luciferases (open reading frames) areshown in bold. The ATG start codons as well as the TAA stop codons areunderlined.

In addition to the nucleotidic cDNA sequences, and the respectiveamino-acid sequences of the proteins, the two relevant properties thatcharacterize the biological activity of these proteins (luciferases),are the catalysis of bioluminescence of distinct colors through theoxidation of the same substrate, the firefly D-luciferin[D-2-(6′hydroxy-2′benzothiaxolyl)Δ²-thiazoline-4-carboxylic acid], asfollows:

-   -   luciferase coded by Phrixothrix vivianii cDNA which produce        green light (with maximum of electromagnetic spectrum centered        at λ_(max)=549 nm) luciferase coded by Phrixothrix hirtus cDNA        which produce red light (with maximum of electromagnetic        spectrum centered at λ_(max)=622 nm; other cloned luciferases        emit in the range between λ_(max)=546-593 nm of the spectrum).        Comparison of Phrixothrix Luciferases.

Although a Phengodes luciferase had already been cloned, the sequencesof Phrixothrix are the first ones of Phengodidae to be reported. Theidentity between Phrixothrix luciferases (71%) is lower than thatexpected for proteins of different species of the same genus, usuallyabove 80% in the case of click-beetle (16) and of firefly (11, 13)luciferases. Some dissimilarity must result from the divergence of theseluciferases toward the emission of distinct bioluminescence colors forthe distinct biological functions played by the lateral and headlanterns. Indeed, the luciferase of R. ohbai railroad-worm, which arisesfrom lateral lanterns with function and bioluminescence colors similarto those of P. vivianii railroad-worm lateral lanterns (37), showedhigher identity with Pv_(GR) luciferase (66%) than with Ph_(RE)luciferase (56%) that emits a color very different from that of adistinct lantern.

Comparison of Phrixothrix luciferases with a set of 12 other beetleluciferases showed 118 invariant residues, most of them located in theC-terminal region. Beside these invariant residues, Phrixothrixluciferases showed 21 additional residues in common with click-beetleluciferases and 31 residues with firefly luciferases. Most of theresidues in common with click-beetle luciferases are located in theregion between residues 242 and 333, whereas those in common withfirefly luciferases are preferentially located in the region betweenresidues 310 and 350. The peroxisomal targeting tripeptide SKL was foundin all railroad-worm and click-beetle luciferases and in most fireflyluciferases.

Residues Typical of Ph_(RE) Luciferase.

The region from residue 300 to about 480 appears to be more homologouswith Pv_(GR) and R. ohbai green light-emitting luciferases than withPh_(RE) luciferase. In Ph_(RE) luciferase, the occurrence of many uniquesubstitutions in this otherwise conserved region suggests that thisregion plays some role in the bioluminescence color determination. Thesubstitution of Ala314 by Ser in Ph_(RE) luciferase is located in afragment which supposedly interacts with luciferin according to arecently proposed model (42). Whereas substitution of Ala 314 by Ser isassociated with considerable chemical changes, the substitution ofIle410 by Leu (Ph_(RE) numbering), except for sterical hindranceeffects, does not seem to have considerable influence since theseresidues have very similar chemical properties. Other substitutions liein the region 430-480, mainly in the fragment 469-479 which displays aquite hydrophilic character in relation to other luciferases. Inclick-beetle isoenzymes, which emit such different colors as green andorange, this fragment is invariable and thus does not account for colordetermination. If such substitutions influence bioluminescence spectrathrough a cumulative effect, it is more likely that they do so throughinfluencing the conformation of active site, rather than through asolvent effect, since no trend indicative of a relationship betweenhydropathy profiles and bioluminescence colors was found. Furthermore,the solvent effect created by many substitutions on the active site,although influencing short-range spectral shifts (<40 nm), is not enoughonly by itself to explain green-red shift, since fluorescence studies onoxyluciferin, dehydroluciferin, and analogues, in solvents with distinctdielectric constants, failed to get such a large spectral shift as thatobserved in Phrixothrix luciferases (>7 nm) (45, 46).

The presence of Arg352 in Ph_(RE) luciferase, which corresponds to thedeleted residue in the shorter Pv_(GR) luciferase, is another importantdistinctive structural feature between these proteins. In Ph_(RE) thisregion (residues 350-362) is associated with a large increase ofhydrophobicity in relation to Pv_(GR) luciferase. Such a feature couldbe potentially involved with considerable conformational changes amongthese luciferases. Such changes could affect the bioluminescence colorsthrough proper positioning a basic residue in the neighbors of C-5 ofexcited oxyluciferin in the case of yellow-green-emitting luciferases(20), or by influencing the active site geometry according to hypothesisIII (23).

Comparison of Phrixothrix Luciferases with Firefly Luciferase RedMutants.

Other residues whose mutation in firefly luciferases results in redlight (26-28) were compared with Ph_(RE) and Pv_(GR) luciferases.Ser286, whose substitution by Asn in the firefly luciferase results in ared shift (26), was found to be conserved in Ph_(RE) luciferase, butreplaced by Thr in Pv_(GR) and R. ohbai luciferases. Because Ser and Thrhave very similar properties, these distinct residues do not seem to berelated with determination of bioluminescence color in Phrixothrixluciferases. Gly 326, which is replaced by Ser in the firefly red mutantCM-2 (26), was found to be conserved among firefly luciferases, but itwas replaced by Ala in all railroad-worm and click-beetle luciferasesstudied. Thus these substitutions cannot account for red bioluminescencein Ph_(RE) such as in the case of firefly luciferase mutants, probablybecause changes at these positions affect differently the tridimensionalstructure of these luciferases. Indeed, the substitutions in the redmutants always involved replacements of charged or polar groups, whichcan potentially interact with other residues in the tertiary structureof the firefly luciferase through electrostatic interactions or byhydrogen bonding, to keep a conformation necessary for yellow-greenlight emission. Also, no particular similarities were found betweenPh_(RE) luciferase and P. plagiophthalamus orange light-emittingisoenzyme, which emits closer bioluminescence color (λ_(max)=593 nm).Ph_(RE) and Pv_(GR) luciferases showed independently the same degree ofhomology with both P. plagiophthalamus green and orangebioluminescence-emitting isoenzymes.

Nucleic Acids of the Invention.

In view of the foregoing, the invention pertains to isolated nucleicacid molecules comprising nucleotide sequences encoding railroad wormluciferases, and the complement of these isolated nucleic acidmolecules. The nucleic acid molecules can be double-stranded orsingle-stranded; single stranded nucleic acid molecules can be eitherthe coding (sense) strand or the non-coding (antisense) strand. Thenucleic acid molecules can additionally contain a marker sequence, forexample, a nucleotide sequence which encodes a polypeptide, to assist inisolation or purification of the polypeptide. Such sequences include,but are not limited to, those which encode a glutathione-S-transferase(GST) fusion protein and those which encode a hemaglutinin A (HA)peptide marker from influenza. In a preferred embodiment, the nucleicacid molecule has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

As used herein, an “isolated” or “substantially pure” gene or nucleicacid molecule is intended to mean a gene which is not flanked bynucleotide sequences which normally (in nature) flank the gene (as inother genomic sequences). Thus, an isolated gene can include a genewhich is synthesized chemically or by recombinant means. Thus,recombinant DNA contained in a vector are included in the definition of“isolated” as used herein. Also, isolated nucleotide sequences includerecombinant DNA molecules in heterologous host cells, as well aspartially or substantially purified DNA molecules in solution. Suchisolated nucleotide sequences are useful in the manufacture of theencoded protein, as probes for isolating homologous sequences (e.g.,from other mammalian species), for gene mapping (e.g., by in situhybridization with chromosomes), or for detecting expression of theluciferase gene in tissue (e.g., human tissue), such as by Northern blotanalysis.

The present invention also encompasses variations of the nucleic acidsequences of the invention. Such variations can be naturally-occurring,such as in the case of allelic variation, or non-naturally-occurring,such as those induced by various mutagens and mutagenic processes.Intended variations include, but are not limited to, addition, deletionand substitution of one or more nucleotides which can result inconservative or non-conservative amino acid changes, including additionsand deletions. Preferably, the nucleotide or amino acid variations aresilent or conserved; that is, they do not alter the characteristics oractivity of the railroad-worm luciferases described herein.

Other alterations of the nucleic acid molecules of the invention caninclude, for example, labeling, methylation, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g.,phosphorothioates, phosphorodithioates), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen), chelators,alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).Also included are synthetic molecules that mimic nucleic acid moleculesin the ability to bind to a designated sequences via hydrogen bondingand other chemical interactions. Such molecules include, for example,those in which peptide linkages substitute for phosphate linkages in thebackbone of the molecule.

The invention also relates to fragments of the isolated nucleic acidmolecules described herein. The term “fragment” is intended to encompassa portion of a nucleic acid sequence described herein which is from atleast about 25 contiguous nucleotides to at least about 50 contiguousnucleotides or longer in length. One or more introns can also bepresent. Such fragments are useful as probes, e.g., for diagnosticmethods, as described below and also as primers or probes. Particularlypreferred primers and probes selectively hybridize to a nucleic acidmolecule containing the luciferase genes described herein.

The invention also pertains to nucleic acid molecules which hybridizeunder high stringency hybridization conditions, such as for selectivehybridization, to a nucleotide sequence described herein (e.g., nucleicacid molecules which specifically hybridize to a nucleic acid containingthe luciferase genes described herein). Hybridization probes areoligonucleotides which bind in a base-specific manner to a complementarystrand of nucleic acid. Suitable probes include polypeptide nucleicacids, as described in (Nielsen et al., Science 254:1497-1500 (1991)).

Such nucleic acid molecules can be detected and/or isolated by specifichybridization (e.g., under high stringency conditions). “Stringencyconditions” for hybridization is a term of art which refers to theincubation and wash conditions, e.g., conditions of temperature andbuffer concentration, which permit hybridization of a particular nucleicacid to a second nucleic acid; the first nucleic acid may be perfectly(i.e., 100%) complementary to the second, or the first and second mayshare some degree of complementarity which is less than perfect (e.g.,60%, 75%, 85%, 95%). For example, certain high stringency conditions canbe used which distinguish perfectly complementary nucleic acids fromthose of less complementarity.

“High stringency conditions”, “moderate stringency conditions” and “lowstringency conditions” for nucleic acid hybridizations are explained onpages 2.10.1-2.10.16 and pages 6.3.1-6 in Current Protocols in MolecularBiology (Ausubel, F. M. et al., “Current Protocols in MolecularBiology”, John Wiley & Sons, (1998)) the teachings of which are herebyincorporated by reference. The exact conditions which determine thestringency of hybridization depend not only on ionic strength (e.g.,0.2×SSC, 0.1×SSC), temperature (e.g., room temperature, 42° C., 68° C.)and the concentration of destabilizing agents such as formamide ordenaturing agents such as SDS, but also on factors such as the length ofthe nucleic acid sequence, base composition, percent mismatch betweenhybridizing sequences and the frequency of occurrence of subsets of thatsequence within other non-identical sequences. Thus, high, moderate orlow stringency conditions can be determined empirically. By varyinghybridization conditions from a level of stringency at which nohybridization occurs to a level at which hybridization is firstobserved, conditions which will allow a given sequence to hybridize(e.g., selectively) with the most similar sequences in the sample can bedetermined.

Exemplary conditions are described in Krause, M. H. and S. A. Aaronson,Methods in Enzymology, 200:546-556 (1991). Also, in, Ausubel, et al.,“Current Protocols in Molecular Biology”, John Wiley & Sons, (1998),which describes the determination of washing conditions for moderate orlow stringency conditions. Washing is the step in which conditions areusually set so as to determine a minimum level of complementarity of thehybrids. Generally, starting from the lowest temperature at which onlyhomologous hybridization occurs, each ° C. by which the final washtemperature is reduced (holding SSC concentration constant) allows anincrease by 1% in the maximum extent of mismatching among the sequencesthat hybridize. Generally, doubling the concentration of SSC results inan increase in T_(m) of ˜17° C. Using these guidelines, the washingtemperature can be determined empirically for high, moderate or lowstringency, depending on the level of mismatch sought.

For example, a low stringency wash can comprise washing in a solutioncontaining 0.2×SSC/0.1% SDS for 10 min at room temperature; a moderatestringency wash can comprise washing in a prewarmed solution (42° C.)solution containing 0.2×SSC/0.1% SDS for 15 min at 42° C.; and a highstringency wash can comprise washing in prewarmed (68° C.) solutioncontaining 0.1×SSC/0.1% SDS for 15 min at 68° C. Furthermore, washes canbe performed repeatedly or sequentially to obtain a desired result asknown in the art. Equivalent conditions can be determined by varying oneor more of the parameters given as an example, as known in the art,while maintaining a similar degree of identity or similarity between thetarget nucleic acid molecule and the primer or probe used.

Hybridizable nucleic acid molecules are useful as probes and primers,e.g., for diagnostic applications, as described below. As used herein,the term “primer” refers to a single-stranded oligonucleotide which actsas a point of initiation of template-directed DNA synthesis underappropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as, DNAor RNA polymerase or reverse transcriptase) in an appropriate buffer andat a suitable temperature. The appropriate length of a primer depends onthe intended use of the primer, but typically ranges from 15 to 30nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the template,but must be sufficiently complementary to hybridize with a template. Theterm “primer site” refers to the area of the target DNA to which aprimer hybridizes. The term “primer pair” refers to a set of primersincluding a 5′ (upstream) primer that hybridizes with the 5′ end of theDNA sequence to be amplified and a 3′ (downstream) primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

The invention also pertains to nucleotide sequences which have asubstantial identity with the nucleotide sequences described herein;particularly preferred are nucleotide sequences which have at leastabout 70%, and more preferably at least about 80% identity, and evenmore preferably at least about 90% identity, with nucleotide sequencesdescribed herein. Particularly preferred in this instance are nucleotidesequences encoding railroad-worm luciferases.

To determine the percent identity of two nucleotide sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of a first nucleotide sequence). Thenucleotides at corresponding nucleotide positions are then compared.When a position in the first sequence is occupied by the same nucleotideas the corresponding position in the second sequence, then the moleculesare identical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin et al. (Proc. Natl. Acad. Sci. USA,90:5873-5877 (1993)). Such an algorithm is incorporated into the NBLASTprogram which can be used to identify sequences having the desiredidentity to nucleotide sequences of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (Nucleic Acids Res, 25:3389-3402 (1997)).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., NBLAST) can be used. In oneembodiment, parameters for sequence comparison can be set at W=12.Parameters can also be varied (e.g., W=5 or W=20). The value “W”determines how many continuous nucleotides must be identical for theprogram to identify two sequences as containing regions of identity.

In a related aspect, the nucleic acid fragments of the invention areused as probes or primers in assays such as those described herein.“Probes” are oligonucleotides that hybridize in a base-specific mannerto a complementary strand of nucleic acid molecules. Such probes includepolypeptide nucleic acids, as described in Nielsen et al., Science, 254,1497-1500 (1991). Typically, a probe comprises a region of nueleotidesequence that hybridizes-under highly stringent conditions to at leastabout 15, typically about 20-25, and more typically about 40, 50 or 75,consecutive nucleotides of a nucleic acid molecule comprising anucleotide sequence selected from SEQ ID NOs: 1 or 3, the complement ofSEQ ID NOs: 1 or 3. More typically, the probe further comprises a label,e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.

The invention also provides expression vectors containing a nucleic acidcomprising the luciferase genes, operatively linked to at least oneregulatory sequence. Many such vectors are commercially available, andother suitable vectors can be readily prepared by the skilled artisan.“Operatively linked” is intended to mean that the nucleic acid sequenceis linked to a regulatory sequence in a manner which allows expressionof the nucleic acid sequence. Regulatory sequences are art-recognizedand are selected to produce the luciferases described herein.Accordingly, the term “regulatory sequence” includes promoters,enhancers, and other expression control elements such as those describedin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990). For example, the nativeregulatory sequences or regulatory sequences native to the transformedhost cell can be employed. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the receptor desired to be expressed.For instance, the gene of the present invention can be expressed byligating the gene into a vector suitable for expression in eitherprokaryotic cells, eukaryotic cells or both (see, for example, Broach,et al., Experimental Manipulation of Gene Expression, ed. M. Inouye(Academic Press, 1983) p. 83; Molecular Cloning: A Laboratory Manual,2nd Ed., ed. Sambrook et al. (Cold Spring Harbor Laboratory Press, 1989)Chapters 16 and 17). Typically, expression constructs will contain oneor more selectable markers, including, but not limited to, the gene thatencodes dihydrofolate reductase and the genes that confer resistance toneomycin, tetracycline, ampicillin, chloramphenicol, kanamycin andstreptomycin resistance. Vectors can also include, for example, anautonomously replicating sequence (ARS), expression control sequences,ribosome-binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, secretion signals and mRNAstabilizing sequences.

Prokaryotic and eukaryotic host cells transformed by the describedvectors are also provided by this invention. For instance, cells whichcan be transformed with the vectors of the present invention include,but are not limited to, bacterial cells such as E. coli (e.g., E. coliK12 strains), Streptomyces, Pseudomonas, Serratia marcescens andSalmonella typhimurium, insect cells (baculovirus), includingDrosophila, fungal cells, such as yeast cells, plant cells and mammaliancells, such as thymocytes, Chinese hamster ovary cells (CHO), and COScells. The host cells can be transformed by the described vectors byvarious methods (e.g., electroporation, transfection using calciumchloride, rubidium chloride, calcium phosphate, DEAE-dextran, or othersubstances; microprojectile bombardment; lipofection, infection wherethe vector is an infectious agent such as a retroviral genome, and othermethods), depending on the type of cellular host.

The nucleic acid molecules of the present invention can be produced, forexample, by replication in a suitable host cell, as described above.Alternatively, the nucleic acid molecules can also be produced bychemical synthesis.

The nucleotide sequences of the nucleic acid molecules described herein(e.g., a nucleic acid molecule comprising SEQ ID NO:1 or SEQ ID NO:3)can be amplified by methods known in the art. For example, this can beaccomplished by e.g., PCR. See generally PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. No. 4,683,202.

Other suitable amplification methods include the ligase chain reaction(LCR) (see Wu and Wallace, Genomics 4:560 (1989), Landegren et al.,Science 241:1077 (1988), transcription amplification (Kwoh et al., Proc.Natl. Acad. Sci. USA 86:1173 (1989)), and self-sustained sequencereplication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990))and nucleic acid based sequence amplification (NASBA). The latter twoamplification methods involve isothermal reactions based on isothermaltranscription, which produce both single stranded RNA (ssRNA) and doublestranded DNA (dsDNA) as the amplification products in a ratio of about30 or 100 to 1, respectively.

The amplified DNA can be radiolabeled and used as a probe for screeninga library or other suitable vector to identify homologous nucleotidesequences. Corresponding clones can be isolated, DNA can be obtainedfollowing in vivo excision, and the cloned insert can be sequenced ineither or both orientations by art recognized methods, to identify thecorrect reading frame encoding a protein of the appropriate molecularweight. For example, the direct analysis of the nucleotide sequence ofhomologous nucleic acid molecules of the present invention can beaccomplished using either the dideoxy chain termination method or theMaxam Gilbert method (see Sambrook et al., Molecular Cloning, ALaboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al.,Recombinant DNA Laboratory Manual, (Acad. Press, 1988)). Using these orsimilar methods, the protein(s) and the DNA encoding the protein can beisolated, sequenced and further characterized.

Antisense nucleic acid molecules of the invention can be designed usingthe nucleotide sequences of the present invention and the complementsthereof, and constructed using chemical synthesis and enzymatic ligationreactions using procedures known in the art. For example, an antisensenucleic acid molecule (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Alternatively, the antisense nucleic acid molecule can be producedbiologically using an expression vector into which a nucleic acidmolecule has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid molecule will be of anantisense orientation to a target nucleic acid of interest).

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into which anucleic acid molecule of the invention has been introduced. Such hostcells can then be used to create non-human transgenic animals in whichexogenous nucleotide sequences have been introduced into the genome orhomologous recombinant animals in which endogenous nucleotide sequenceshave been altered. Such animals are useful for studying the functionand/or activity of the nucleotide sequence and polypeptide encoded bythe sequence and for identifying and/or evaluating modulators of theiractivity. As used herein, a “transgenic animal” is a non-human animal,preferably a mammal, more preferably a rodent such as a rat or mouse, inwhich one or more of the cells of the animal includes a transgene. Otherexamples of transgenic animals include non-human primates, sheep, dogs,cows, goats, chickens and amphibians. A transgene is exogenous DNA whichis integrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, an“homologous recombinant animal” is a non-human animal, preferably amammal, more preferably a mouse, in which an endogenous gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986). Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley (1991) Current Opinion in Bio/Technology, 2:823-829and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO93/04169. Clones of the non-human transgenic animals described hereincan also be produced according to the methods described in Wilmut et al.(1997) Nature, 385:810-813 and PCT Publication Nos. WO 97/07668 and WO97/07669.

Peptides and Proteins of the Invention

The present invention also provides isolated polypeptides and variantsand fragments thereof that are encoded by the nucleic acid molecules ofthe invention. For example, as described above, the nucleotide sequencescan be used to design primers to clone and express cDNAs encoding thepolypeptides of the invention.

As used herein, a polypeptide is said to be “isolated” or “purified”when it is substantially free of cellular material when it is isolatedfrom recombinant and non-recombinant cells, or free of chemicalprecursors or other chemicals when it is chemically synthesized. Apolypeptide, however, can be joined to another polypeptide with which itis not normally associated in a cell and still be “isolated” or“purified.”

The polypeptides of the invention can be purified to homogeneity. It isunderstood, however, that preparations in which the polypeptide is notpurified to homogeneity are useful. The critical feature is that thepreparation allows for the desired function of the polypeptide, even inthe presence of considerable amounts of other components. Thus, theinvention encompasses various degrees of purity. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof the polypeptide having less than about 30% (by dry weight) otherproteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins.

When a polypeptide is recombinantly produced, it can also besubstantially free of culture medium, i.e., culture medium representsless than about 20%, less than about 10%, or less than about 5% of thevolume of the protein preparation. The language “substantially free ofchemical precursors or other chemicals” includes preparations of thepolypeptide in which it is separated from chemical precursors or otherchemicals that are involved in its synthesis. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of the polypeptide having less than about 30% (bydry weight) chemical precursors or other chemicals, less than about 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

In one embodiment, a polypeptide comprises an amino acid sequenceencoded by a nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 andcomplements and portions thereof. However, the invention alsoencompasses sequence variants. Variants include a substantiallyhomologous protein encoded by the same genetic locus in an organism,i.e., an allelic variant. Variants also encompass proteins derived fromother genetic loci in an organism, but having substantial homology to apolypeptide encoded by a nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3 and complements and portions thereof. Variants also include proteinssubstantially homologous or identical to these polypeptides but derivedfrom another organism, i.e., an ortholog. Variants also include proteinsthat are substantially homologous or identical to these polypeptidesthat are produced by chemical synthesis. Variants also include proteinsthat are substantially homologous or identical to these polypeptidesthat are produced by recombinant methods.

As used herein, two proteins (or a region of the proteins) aresubstantially homologous or identical when the amino acid sequences areat least about 45-55%, typically at least about 70-75%, more typicallyat least about 80-85%, and most typically greater than about 93% or morehomologous or identical. A substantially homologous amino acid sequence,according to the present invention, will be encoded by a nucleic acidmolecule hybridizing to SEQ ID NO: 1, SEQ ID NO: 3 or portion thereof,under stringent conditions as more particularly described above.

To determine the percent homology or identity of two amino acidsequences, or of two nucleic acid sequences, the sequences are alignedfor optimal comparison purposes (e.g., gaps can be introduced in thesequence of one protein or nucleic acid molecule for optimal alignmentwith the other protein or nucleic acid molecule). The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in one sequenceis occupied by the same amino acid residue or nucleotide as thecorresponding position in the other sequence, then the molecules arehomologous at that position. As used herein, amino acid or nucleic acid“homology” is equivalent to amino acid or nucleic acid “identity”. Thepercent homology between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., percent homologyequals the number of identical positions/total number of positions times100).

The invention also encompasses polypeptides having a lower degree ofidentity but having sufficient similarity so as to perform one or moreof the same functions performed by a polypeptide encoded by a nucleicacid molecule of the invention. Similarity is determined by conservedamino acid substitution. Such substitutions are those that substitute agiven amino acid in a polypeptide by another amino acid of likecharacteristics. Conservative substitutions are likely to bephenotypically silent. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990).

A variant polypeptide can differ in amino acid sequence by one or moresubstitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these. Further, variantpolypeptides can be fully functional or can lack function in one or moreactivities. Fully functional variants typically contain onlyconservative variation or variation in non-critical residues or innon-critical regions. Functional variants can also contain substitutionof similar amino acids that result in no change or an insignificantchange in function. Alternatively, such substitutions may positively ornegatively affect function to some degree. Non-functional variantstypically contain one or more non-conservative amino acid substitutions,deletions, insertions, inversions, or truncation or a substitution,insertion, inversion, or deletion in a critical residue or criticalregion.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science, 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity in vitro, or in vitro proliferative activity. Sitesthat are critical for polypeptide activity can also be determined bystructural analysis such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith et al., J. Mol. Biol., 224:899-904(1992); de Vos et al. Science, 255:306-312 (1992)).

The invention also includes polypeptide fragments of the polypeptides ofthe invention. Fragments can be derived from a polypeptide encoded by anucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO: 3 or a portionthereof and the complements thereof. However, the invention alsoencompasses fragments of the variants of the polypeptides describedherein. As used herein, a fragment comprises at least 6 contiguous aminoacids. Useful fragments include those that retain one or more of thebiological activities of the polypeptide as well as fragments that canbe used as an immunogen to generate polypeptide-specific antibodies.

Biologically active fragments (peptides which are, for example, 6, 9,12, 15, 16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acidsin length) can comprise a domain, segment, or motif that has beenidentified by analysis of the polypeptide sequence using well-knownmethods, e.g., signal peptides, extracellular domains, one or moretransmembrane segments or loops, ligand binding regions, zinc fingerdomains, DNA binding domains, acylation sites, glycosylation sites, orphosphorylation sites.

Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Further, severalfragments can be comprised within a single larger polypeptide. In oneembodiment a fragment designed for expression in a host can haveheterologous pre- and pro-polypeptide regions fused to the aminoterminus of the polypeptide fragment and an additional region fused tothe carboxyl terminus of the fragment.

The invention thus provides chimeric or fusion proteins. These comprisea polypeptide of the invention operatively linked to a heterologousprotein having an amino acid sequence not substantially homologous tothe polypeptide. “Operatively linked” indicates that the polypeptideprotein and the heterologous protein are fused in-frame. Theheterologous protein can be fused to the N-terminus or C-terminus of thepolypeptide. In one embodiment the fusion protein does not affectfunction of the polypeptid per se. For example, the fusion protein canbe a GST-fusion protein in which the polypeptide sequences are fused tothe C-terminus of the GST sequences. Other types of fusion proteinsinclude, but are not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions and Ig fusions. Such fusion proteins, particularly poly-Hisfusions, can facilitate the purification of recombinant polypeptide. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of a protein can be increased by using a heterologous signalsequence. Therefore, in another embodiment, the fusion protein containsa heterologous signal sequence at its N-terminus.

EP-A-O 464 533 discloses fusion proteins comprising various portions ofimmunoglobulin constant regions. The Fc is useful in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). In drug discovery, for example, humanproteins have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists. Bennett etal., Journal of Molecular Recognition, 8:52-58 (1995) and Johanson etal., The Journal of Biological Chemistry, 270, 16:9459-9471 (1995).Thus, this invention also encompasses soluble fusion proteins containinga polypeptide of the invention and various portions of the constantregions of heavy or light chains of immunoglobulins of various subclass(IgG, IgM, IgA, IgE).

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of nucleic acid fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive nucleic acid fragments which cansubsequently be annealed and re-amplified to generate a chimeric nucleicacid sequence (see Ausubel et al., Current Protocols in MolecularBiology, 1992). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST protein). Anucleic acid molecule encoding a polypeptide of the invention can becloned into such an expression vector such that the fusion moiety islinked in-frame to the polypeptide protein.

The isolated polypeptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods. Inone embodiment, the protein is produced by recombinant DNA techniques.For example, a nucleic acid molecule encoding the polypeptide is clonedinto an expression vector, the expression vector introduced into a hostcell and the protein expressed in the host cell. The protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques.

In general, polypeptides or proteins of the present invention can beused as a molecular weight marker on SDS-PAGE gels or on molecular sievegel filtration columns using art-recognized methods. The polypeptides ofthe present invention can be used to raise antibodies or to elicit animmune response. The polypeptides can also be used as a reagent, e.g., alabeled reagent, in assays to quantitatively determine levels of theprotein or a molecule to which it binds (e.g., a receptor or a ligand)in biological fluids. The polypeptides can also be used as markers forcells or tissues in which the corresponding protein is preferentiallyexpressed, either constitutively, during tissue differentiation, or in adiseased state. The polypeptides can be used to isolate a correspondingbinding partner, e.g., receptor or ligand, such as, for example, in aninteraction trap assay, and to screen for peptide or small moleculeantagonists or agonists of the binding interaction.

In another aspect, the invention provides antibodies to the polypeptidesand polypeptide fragments of the invention, e.g., having an amino acidsequence encoded by a nucleic acid molecule comprising all or a portionof SEQ ID NO: 1 or SEQ ID NO: 3. The term “antibody” as used hereinrefers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds an antigen. A molecule thatspecifically binds to a polypeptide of the invention is a molecule thatbinds to that polypeptide or a fragment thereof, but does notsubstantially bind other molecules in a sample, e.g., a biologicalsample, which naturally contains the polypeptide. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind to a polypeptide of theinvention. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of a polypeptide ofthe invention. A monoclonal antibody composition thus typically displaysa single binding affinity for a particular polypeptide of the inventionwith which it immunoreacts.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a desired immunogen, e.g., polypeptide of theinvention or fragment thereof. The antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilizedpolypeptide. If desired, the antibody molecules directed against thepolypeptide can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature, 256:495-497, the human B cell hybridoma technique (Kozbor et al.(1983) Immol. Today, 4:72), the EBV-hybridoma technique (Cole et al.(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96) or trioma techniques. The technology for producing hybridomasis well known (see generally Current Protocols in Immunology (1994)Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly,an immortal cell line (typically a myeloma) is fused to lymphocytes(typically splenocytes) from a mammal immunized with an immunogen asdescribed above, and the culture supernatants of the resulting hybridomacells are screened to identify a hybridoma producing a monoclonalantibody that binds a polypeptide of the invention.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody to a polypeptide of the invention (see, e.g.,Current Protocols in Immunology, supra; Galfre et al. (1977) Nature,266:55052; R. H. Kenneth, in Monoclonal Antibodies: A New Dimension InBiological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); andLerner (1981) Yale J. Biol. Med., 54:387-402. Moreover, the ordinarilyskilled worker will appreciate that there are many variations of suchmethods that also would be useful.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody to a polypeptide of the invention can be identifiedand isolated by screening a recombinant combinatorial immunoglobulinlibrary (e.g., an antibody phage display library) with the polypeptideto thereby isolate immunoglobulin library members that bind thepolypeptide. Kits for generating and screening phage display librariesare commercially available (e.g., the Pharmacia Recombinant PhageAntibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™Phage Display Kit, Catalog No. 240612). Additionally, examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, U.S.Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO90/02809; Fuchs et al. (1991) Bio/Technology, 9:1370-1372; Hay et al.(1992) Hum. Antibod. Hybridomas, 3:81-85; Huse et al. (1989) Science,246:1275-1281; Griffiths et al. (1993) EMBO J., 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart.

In general, antibodies of the invention (e.g., a monoclonal antibody)can be used to isolate a polypeptide of the invention by standardtechniques, such as affinity chromatography or immunoprecipitation. Apolypeptide-specific antibody can facilitate the purification of naturalpolypeptide from cells and of recombinantly produced polypeptideexpressed in host cells. Moreover, an antibody specific for apolypeptide of the invention can be used to detect the polypeptide(e.g., in a cellular lysate, cell supernatant, or tissue sample) inorder to evaluate the abundance and pattern of expression of thepolypeptide. Antibodies can be used diagnostically to monitor proteinlevels in tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen. Detectioncan be facilitated by coupling the antibody to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude 125I, 131I, 35S or 3H.

Applicability.

Nucleic acid molecules of this invention can be used as sensitive andversatile tools to report molecular events genetically regulated insideliving cells and their extracts in biological assays. (See Gould andSubramani, 1988). The luciferase genes can be ligated to DNA vectors(such as plasmids and virus) downstream regulator elements calledpromoters, and inserted inside the cells. Under the proper conditions,the promoter will drive the transcription of the luciferase gene and theexpression of active luciferases in a manner that is dependent of thedegree of activation of the promoter. The expression of the luciferaseis directly proportional to the amount of light produced in presence ofexogenously supplied D-luciferin and ATP (present in the intracellularmedium) and can be quantified using commercially available photometers,such as luminometers. Based on this principle, luciferase genes can beused to monitor the transcriptional activity of promoters, to diagnosticand quantify the spreading of viral infections in animal and vegetaltissues or to diagnostic the presence of virus in biological samples. Inthis case, the luciferase gene is ligated to a DNA vector downstream aviral promoter, which drives the transcription and expression ofluciferase. This system is then introduced inside the cell or thebiological sample to be tested. If the biological sample is infected bythe virus from which the promoter was isolated, the molecular signalsresulting from such virus will activate the promoter that will start totranscribe the luciferase gene and to express luciferase, whose lightwill confirm the infection. In addition, the amount of light producedwill be proportional to the degree of promoter activation which can beused to quantify the titer of the infection (this methodology wasalready successfully used to detect the presence of virus such as HIV).In brief, luciferase genes can be used as reporter genes for thefollowing purposes: (1) promoter transcriptional activity determination(e.g., single and dual promoters); (2) viral diagnostics andquantification; (3) in vivo expression and visualization in (through)mammalian tissues such as blood, bone tissue and muscular tissue; (3)cytotoxcicity tests; (4) cell viability determination; (5) ATPquantification in biological samples; (6) biosensor for environmentaland chemical stress agents, among others. See Gould and Subramani, 1988;Schwartz et al., 1990; Contag et al., 1995; Comhaire et al., 1989;Stanley, 1989; Lundin et al., 1989; Tauriainen et al., 1999.

In the past, only the gene for the luciferase of the North-Americanfirefly Photinus pyralis, which produces yellow-green light, has beenwidely applied as reporter gene. One disadvantage of this reporter geneis that the yellow-green light is considerably absorbed in certainbiological samples with high optical density in the blue-yellow regionof the spectrum such as the blood and other highly pigmented biologicalsamples. Other genes that code for beetle luciferases that emit in thegreen-orange region of the spectrum were also cloned (Wood, 1995), butthey were not widely used and they suffer similar limitations inpigmented biological samples.

The gene of the red emitting luciferase arising from Phrixothrixdescribed herein (Viviani et al., 1999; incorporated herein in itsentirety) offer a notable wider potential for application, mainly forpurposes of detecting light in biological samples with high opticaldensity in the blue-yellow region of the spectrum such as blood, bonetissue and muscular tissue among others. In addition, the simultaneoususe of the genes for the green and red emitting luciferases, with theaim to monitor two different promoters (regulator elements) at the sametime, offer the possibility to use 2 reporter genes whose signals can beclearly distinguished by the color of the light, with a minimum ofbioluminescence spectrum overlap (<10%) avoiding interferences.Furthermore, both luciferases, arising from the same-bioluminescentsystem, work under the same-biochemical conditions (in presence of ATP,magnesium ions which are natural intracellular components) andD-Luciferin which should be supplied exogenously. Since the beetleluciferases genes presently used code for luciferases which producelight in the green-orange range of the spectrum, their simultaneousapplication is limited by the considerable degree of emission spectraoverlapping (>25%), imposing severe difficulties to discriminate onesignal from another. One solution to this problem was previouslypatented by Promega Co. (USA) (Sherf et al., 1996), which used the genesfor firefly luciferase which emit yellow-green light and a celenterate(jellyfish) luciferase which emit blue light. However, since theseluciferases arise from different bioluminescent systems, they work indistinct biochemical conditions, which is not always a convenientcondition.

Thus, the research for efficiency is satisfied by the use of theluciferases coded by the sequences object of this patent application,since they are able to produce the same green and red bioluminescence ofPhrixothrix, constituting a system which works in the same biochemicalconditions and whose bioluminescence spectra have not overlapping.

These genes can also be used for the industrial scale production ofluciferases for analytical applications. Light emitted in bioluminescentassays using luciferases is actually detectable by a range ofcommercially avaliable photometric instruments such as luminometers,photon counters, fluorometers, CCD-camera systems.

The invention will be further illustrated by the followingexemplification which is not intended to be limiting in any way.

EXEMPLIFICATIONS

In Vivo Expression of Luciferase By E. Coli

Materials and Methods.

Reagents.

Isopropyl-β-D-thiogalactopyranoside (IPTG¹),5-bromo-4-chloro-3-indoyl-â-galactopyranoside (X-Gal), dithiothreitol(DTT), D-luciferin (sodium salt), ampicillin, tetracyclin, and kanamycinwere from Wako Pure Chemicals (Osaka, Japan); coenzyme-A (CoA) andadenosine triphosphate (ATP) from Oriental Yeast Co (Osaka, Japan);Isogen reagent, restriction enzymes, and Taq polymerase from Nippon Gene(Toyama, Japan); Oligo Tex dT30 and DNA ligation kit from Takara Shuzo(Kyoto, Japan); cDNA synthesis kits from Amersham Pharmacia Biotech(Tokyo, Japan); Gigapack III Gold packaging kit from Stratagene (LaJolla, Calif.); and ABI PRISM Dye terminator Cycle Sequencing kit fromPerkin-Elmer (Foster City, Calif.).

Bacterial Strains and Media.

Echerichia coli XL1-Blue MRF′ and SOLR strains were purchased fromStratagene (La Jolla, Calif.). E. coli cells were usually grown in LuriaBertani (LB) medium (1% bacto-triptone, 0.5% yeast extract, 0.5% NaCl).Cell densities were measured by absorbance at 600 nm.

Insects.

Railroad-worms were collected at night as described (5). Larvae of P.vivianii were collected in pastures at Fazenda São Francisco near ParqueNacional das Emas (prefecture of Mineiros, Goías State). Larvae of P.hirtus were collected into cerradão formation at Fazenda Sta Cruz (CostaRica prefecture, Mato Grosso do Sul State) (31) near the former place.Living specimens were cleaned with distilled water, frozen in liquidnitrogen, and stored at −80° C.

Construction and Screening of cDNA Libraries.

cDNA libraries were constructed using methodology similar to Pyreariniustermitilluminans luciferase cloning (16). For P. vivianii larvae, totalRNA was extracted from the whole bodies (without head) of 8 specimens,yielding 360 μg, using Isogen reagent according established procedures(32). Them RNAs were isolated using Oligo-dT latex in accordance withKuribayashi et al. (33). cDNAs were synthesized from 4 μg of isolatedmRNAs using Time Saver cDNA synthesis kit. The first strand reaction wascarried out in the presence of oligo-dT₁₂₋₁₈ primer. After the synthesisof the second cDNA strand, EcoRI/NotI adaptors were ligated to the bluntended cDNA. The cDNA (about 50 ng for P. vivianii bodies) was ligated to1 μg of EcoRI pre-digested/dephosphorilated λZAP II (Stratagene, LaJolla, Calif.) vector in a volume of 5 μL of ligation reaction mixture(1 mM ATP, 7 mM MgCl2, 1 mM DTT in 50 mM Tris-HCl, pH 8.0, and 1 Weissunit of T4 ligase) overnight at 16° C. The ligation mixtures were thenpackaged using Gigapack III Gold packaging extracts. The originallibrary of P. vivianii bodies (7.5×10⁵ pfu) was then in vivo excisedinto E. coli XL1-Blue cells in the presence of helper phage to obtainpBluescript (pBl) libraries. The excised phagemids were used totransform E. coli SOLR cells. The plasmid library was screened byphotodetection (34) using a cooled-CCD camera system (ATTO; Tokyo,Japan), after spraying 1 mM D-luciferin (0.1 M citrate buffer pH 5.0)onto IPTG induced colonies at 20° C. during 12 h. For P. hirtus headlantern library construction, total RNA was extracted from 17 heads and4 μg of mRNA was used to synthesize cDNA. All other procedures wereessentially similar to those described above, except the originallibrary (2.2×10⁴ pfu) was further amplified (1×10⁹ pfu) before excisionof the pBl library.

Sequence Analysis.

The cDNA for green light-emitting luciferase (Pv_(GR)), the 0.75 kb longEcoRV/BamHI and BamHI/BamHI fragments were subcloned into pBl and pUCvectors, respectively (35). The cDNA for red light-emitting luciferase(Ph_(RE)) was digested with EcoRI, and the 3 resulting fragments (0.8;0.6; 0.3 kbp) were subcloned into EcoRI digested/dephosphorilated pUC 18vector. All of these constructions and the original luciferasecDNA-containing plasmids were sequenced by the dydeoxy chain terminationmethod (36) using dye-labeled terminator kit specifically developed forthe ABI PRISM 377 automatic sequencer (Perkin-Elmer; Foster City-CA).For extension of pUC vector, M₁₃ (−21) and reverse primers-were used,whereas for pBl vector T₇ and reverse primers were used. Threeadditional primers, VA₁ (5′-ATGTACTTTCAATCTCTTTGCTAC-3′) (SEQ ID NO:5),VA₃ (5′-AAGTCTAACTATAAGATAAGTTCTTA-3′) (SEQ ID NO:7), and VA₄(5′-CAAGTTTCAGTTAATCCATAT-3′) (SEQ ID NO:6), were designed from theknown partial sequences in order to sequence the internal regions ofPh_(RE) and Pv_(GR). Sequence comparisons, multi-alignments, anddetermination of the protein hydropathy profiles, molecular weights andisoeletric points were made using version 7.3 of Genetyx-mac software(Software Development Co., Ltd., Tokyo, Japan).

Expression and Preparation of Luciferase Extracts.

Liquid cultures of SOLR cells carrying luciferase insert containing pBlwere grown on LB/amp (50 μg/mL) medium at 37° C. with shaking overnight.The preculture (1/100 vol) was then inoculated in LB/amp (50 μg/mL) inthe presence of 1 mM IPTG and incubated at 23° C. during 24 h(OD₆₀₀=1.8). The cells were harvested by centrifugation at 3000 rpmduring 10 min at 4° C., and the pellet was resuspended in coldextraction buffer (0.1 M sodium phosphate buffer, pH 7.5, containing 2mM EDTA, 1 mM DTT, and 1% Triton X-100). Lysozyme was added to the finalconcentration of 1 mg/mL and the suspension incubated 15 min at 0° C.and frozen at −80° C. during 15 min. The lysate was centrifuged at 12000g at 4° C. during 15 min. The supernatant was then fractionated withammonium sulfate. The fraction precipitated between 55% and 70%saturation was dissolved in cold extraction buffer and stored at −20° C.in the presence of glycerol 15%, to maintain the activity.

Luciferase Assays.

The activity levels were measured using a Luminescencer AB-2000luminometer (Atto; Tokyo, Japan) by integration of total light emitted.The assay consisted of the addition of 50 μL of standard solution (2 mMATP, 0.5 mM D-luciferin, 4 mM MgSO₄ in 0.1 M Tris-HCl buffer, pH 8.0) to10 μL of luciferase-containing extracts at 25° C. In vivo lightintensities were measured after adding 50 μL of 0.5 mM D-luciferin in0.1 M sodium citrate buffer pH 5.0 to 10 μL of bacterial suspension intoa microtiter plate.

Kinetic Measurements.

Measurements of light intensities for K_(M) determinations were madeusing the luminometer described above. For luciferin K_(M) estimation,50 μL of 4 mM ATP solution (0.1 M Tris-HCl, pH 8.0, and 8 mM MgSO₄) wasinjected to 50 μL of crude extract diluted 10 times in 0.1 M Tris-HClbuffer, pH 8.0, containing luciferin (0.03-2 mM). For ATP K_(M)estimation, 50 μL of 0.5 mM luciferin solution (0.1 M Tris-HCl buffer,pH 8.0, 8 mM MgSO₄) was injected to 50 μL of 10 times diluted extractcontaining ATP (0.1-4 mM). The assays were carried out at 25° C. Eachpoint of the Michaelis-Menten curve was assayed in quadruplicate. TheK_(M) values were estimated by Lineweaver-Burk plots of the reciprocalof light intensities versus substrate concentration.

Bioluminescence Spectra.

Emission spectra were determined using a Hitachi F4500spectrofluorometer, supplied with a Hamamatsu Photonics R 928 Fphotomultiplier, with the excitation lamp shut down. The spectra wereautomatically corrected for the photosensitivity of the equipment. Forbacterial in vivo spectra determinations, 500 μL of bacterial suspensionand 500 μL of 0.5 mM D-luciferin in 0.1 M sodium citrate buffer, pH 5.0,and 10 mM MgSO₄ were mixed into a cuvette in front of the emissionwindow (16): In vitro spectra were recorded 3 min after mixing 10-100 μLof luciferase-containing extract to 900 μL of standard reaction mixture(0.5 mM D-luciferin, 2 mM ATP, 4 mM MgSO₄, 0.5 mM CoA, and 1% TritonX-100 in 0.1 M Tris-HCl uffer, pH 8.0), to a final volume of 1 mL, intoa luorometer cuvette (16) in front of the emission window. The pH effecton in vitro spectra was measured in 0.1 M sodium phosphate buffer (pH6-8) instead of Tris-HCl buffer as described (16). The spectra measuredat pH 8.0 in both buffers were essentially identical in shape.

Results

Isolation of Positive Clones and Expression of Active Luciferases.

The P. vivianii body cDNA library yielded 7.5×10⁵ recombinant plaques.After screening about 10,000 colonies, we isolated 1 positive clone forlight emission (Pv_(GR)). The cDNA library for P. hirtus head lanternsyielded 2.2×10⁴ recombinant plaques and was amplified (1×10⁹ pfu) beforeexcision. Four positive clones were found after screening 3000 coloniesof the excised amplified library. The most intense light-emitting clone(Ph_(RE)) was isolated for further analysis. Upon D-luciferin spraying,the IPTG-induced SOLR colonies containing pBl-Pv_(GR) and pBl-Ph_(RE)displayed weak green and red bioluminescence, respectively, visibleafter dark-room eye adaptation. The luminescence maximum intensity ofpBl-Ph_(RE)-containing colonies was reached before and decayed soonerthan that of pBl-Pv_(GR)-containing colonies. Luminometer measurement ofthe total light output of both in in vivo and in vitro bioluminescenceassays of IPTG-induced colonies gave nearly the same values for Pv_(GR)and Ph_(RE) luciferases, after correction for the spectralphotosensitivity of the equipment.

cDNAs Structures and Sequences.

The Pv_(GR) cDNA (NCBI access number: AF139644) is a 1765 bp longfragment. The start codon was found 25 bp downstream from the cDNA 5′terminus, which follows the PstI restriction site of pBluescriptpolylinker. An open reading frame of 1635 bp, which codes for a 545amino acid long polypeptide was found. After the stop codon, a 105 bplong 3′ untranslated region followed by a terminal 26 bp poly-A tail wasfound.

The Ph_(RE) cDNA (NCBI access number: AF139645) is a 1760 bp longfragment. The cDNA has a 41 bp untranslated region upstream from thefirst ATG. The sequence of the last 10 bp before the starting codon wasessentially identical to that of Pv_(GR). An open reading frame of 1638bp, coding for a potential 546 residue long polypeptide was found. Afterthe stop codon, a 61 bp long downstream untranslated region with aterminal 7 bp long poly-A tail was found.

Protein Sequences.

The overall identity between Pv_(GR) and Ph_(RE) luciferases was 71%.Pv_(GR) and Ph_(RE) luciferases showed 66.6% and 56% identity,respectively, with the Japanese railroad-worm Ragophthalmus ohbailuciferase, recently cloned in our laboratory (37). As expected, thesevalues are very close to those observed for Phengodes luciferase, withthe same set of enzymes (17). Pv_(GR) luciferase showed a slightlyhigher identity with firefly luciferases (50-55%) than Ph_(RE) did withthe same set of enzymes (46-49%). Both luciferases showed 47-49%identity with click-beetle luciferases. The overall identity shared withacyl CoA ligases is 25%. Like most beetle luciferases, both luciferasesshowed the peroxissomal targeting tripeptide SKL before the stop codons(7).

Protein Properties.

The calculated molecular weights of Pv_(GR) and Ph_(RE) luciferases were59,626 and 60,951 kDa and were close to those estimated by WesternBlotting (data not shown). The isoeletric points, calculated from thededuced primary structures, were 6.26 and 7.0 for Pv_(GR) and Ph_(RE)luciferases, respectively. Pv_(GR) luciferase showed a small increase inthe proportion of hydrophobic and neutral residue content in relation toPv_(GR) luciferase. The hydropathy profiles of these luciferases weresimilar; however there were some regions which showed major differences.In particular the region from residues 350-360, which includes theadditional Arg residue in Ph_(RE) luciferase, showed a major increase ofhydrophobic character in Ph_(RE) luciferase in relation to Pv_(GR)luciferase.

Kinetic Parameters.

The K_(M) values for ammonium sulfate fractionated extracts weremeasured. For Ph_(RE) luciferase the K_(M) for luciferin was 20 μM,whereas for ATP it was 240 μM. However for unknown reasons Pv_(GR)luciferase K_(M) values were much higher than for Ph_(RE) luciferase(150 μM for luciferin and 350 μM for ATP). The rise time to the peak ofintensity and the decay rate of in vitro bioluminescence reaction werefaster for Ph_(RE) luciferase than for Pv_(GR) (results not shown). Asimilar property was observed for the time course of the in vivobioluminescence after spraying D-luciferin.

Spectral Properties.

The in vivo and in vitro bioluminescence spectra emitted by Pv_(GR)luciferase are centered at 549 nm, thus in the green region, close tothat reported for the native enzyme extracted from the larval P.vivianii lateral lanterns (λ_(max)=542 nm; FIG. 3) (5), and to thespectra emitted by the recently cloned Phengodes (λ_(max)=546 nm) (17)and R. ohbai (λ_(max) 556 nm) (37) luciferases. Both the in vivo and invitro bioluminescence spectra of Ph_(RE) luciferase showed a peak at 622nm (FIG. 3), thus 73 nm shifted in relation to Pv_(GR) luciferase. Thein vitro spectrum is 6 nm blue-shifted in relation to that emitted bythe native enzyme extracted from the laval P. hirtus head lanterns(λ_(max)=628 nm) (5), in part due to distinct equipment used formeasurements. This spectrum is much narrower (half bandwidth=55 nm) thanthat emitted by Pv_(GR) luciferase (half-bandwidth=70.5 nm) and otherbettle luciferases. The bioluminescence spectra of both recombinantluciferases did not suffer red shift upon decreasing the pH from 8.0 to6.0, although they underwent a decrease of intensity.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

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1. An isolated nucleic acid molecule encoding a red light emittingluciferase selected from the group consisting of: a) a nucleic acidmolecule comprising SEQ ID NO:3; and b) a nucleic acid molecule whichhybridizes to SEQ ID NO:3 under high stringency conditions comprisingwashing in a solution containing 0.1×SSC/0.1% SDS for 15 mm at 68° C.,or a nucleic acid molecule that is 100% complementary to the full lengthsequence of a) or b).
 2. A method for the production of red lightemitting polypeptide comprising expressing the isolated nucleic acidmolecule according to claim
 1. 3. A vector comprising the isolatednucleic acid molecule as defined in claim
 1. 4. A recombinant host cellcomprising the vector as defined in claim 3.